The HIV-1 epidemic has resulted in ~5 million new infections in 2004 for a total of 40 million people living with HIV/AIDS. Clinical trials have shown that HIV-1 infection cannot be prevented by immunization with subunits of naturally occurring viral envelope (Env) proteins. However, it is clear from experimental studies that neutralizing antibodies to the Env protein can protect primates from infection. Modified Env proteins that better expose the neutralizing epitopes are now required to accelerate vaccine development. Detailed structural studies of the HIV-1 and SIV Env glycoproteins have revealed that this molecule undergoes substantial changes when it is bound by CD4, which is the initial step of virus-cell interaction. A major objective of HIV vaccine research is to create stable structures that are based on the unliganded form of the trimeric Env molecule. Molecules that resemble a stabilized version of the mature envelope trimer on the virion surface and that better expose existing neutralizing epitopes are currently being investigated. However, the Env structure carried by circulating HIV-1 clearly does not induce broadly neutralizing antibodies in infected individuals. Therefore, whatever the benefits of using a trimeric Env complex as an immunogen for vaccination, the Env protein must still be modified to improve the immunogenicity of conserved neutralizing epitopes. We propose to use directed molecular evolution to accomplish 2 main objectives. In this Phase I SBIR proposal, we will first use in vitro multigene DNA recombination to create libraries of novel chimeric HIV env genes that will be screened to identify a number of stable, cleaved, trimeric forms of the HIV-1 Env ectodomain. Second, upon reaching the goals of the present work, we will evaluate the immunogenicity of these proteins in a subsequent Phase II SBIR proposal. This work will provide a rigorous assessment of the value of trimeric Env molecules as immunogens and will provide lead molecules for further development as vaccine candidates. We are ultimately targeting a commercial vaccine product that will be of value in the prevention of HIV/AIDS. The Specific Aims of this Phase I SBIR proposal are summarized below. They are designed to evaluate the feasibility of creating stable and soluble forms of the HIV-1 Env trimer. -Specific Aim #1: Create libraries of recombined env genes encoding the gp140 ectodomain; -Specific Aim #2: Identify and characterize at least 10 stable forms of fully cleaved Env trimers; -Specific Aim #3: Prepare monomeric gp120 from each of the stable, cleaved Env trimers. [unreadable] [unreadable] [unreadable]